The use of multiple-phase aqueous systems for the extraction of proteins has been disclosed in the prior art. Affinity ligands have been used to increase the selectivity of protein partitioning in these systems. For example, the affinity partitioning of phosphofructokinase from fresh baker's yeast in PEG 6000-bound Cibracron blue F3G-A/dextran is reported in Trends in Biotechnology, Vol. 2, No. 2, 1984, pp. 31-35, "Large-scale affinity purification--state of the art and future prospects." The article notes the high cost of one of the most frequently used polymers, dextran. Similarly, the large-scale purification of formate dehydrogenase from C. boidinii by partitioning with PEG-6000-Procion Red HE3b was reported in "Process Design for Large-Scale Purification of Formate Dehydrogenase from Candida boidinii by Affinity Partition, " Journal of Chromatography, Vol. 376, pp. 375-384, 1986. The feasibility of recycling the PEG-ligand was demonstrated. An enzyme yield of 74% was reported for the largest-scale separation from 220 kg of Candida boidinii cells.
In general, the prior art aqueous two-phase extractions that do not employ affinity ligands have resulted in poor selectivity. On the other hand, the prior art systems that used affinity ligands had very specific applicability and could not be applied to a very wide variety of proteins.
Many of the prior art affinity liquid-liquid extraction systems require the use of dextran, which is expensive. While the use of PEG/salt systems would greatly reduce the expense, high salt concentrations disrupt most affinity interactions. High concentrations of certain salts, however, are known to promote metal binding. In the present invention, the interaction between the protein and the metal held by the PEG-chelate is not disrupted by high concentrations of the salts used to form aqueous two-phase systems. These salts include alkali metal and alkaline earth metal citrates, carbonates, silicates, sulfates, formates, succinates, tartrates and phosphates. They are generally used in concentrations between 5 and 15% (w/w) of the total solution for forming two phases. The concentration appropriate for any specific system is determined by the phase diagram of the particular system.
Metals have been immobilized by chelates attached to resins for purification of proteins by chromatography and fixed-bed adsorption. This was first demonstrated in 1975 by Porath (Nature, Vol. 258, pp. 598-599). Although U.S. Pat. No. 4,765,834 discloses the use of aqueous multiphase systems for the recovery of metals, metal-chelating polymers have not been used for extraction or precipitation of proteins.
There have been some reports of affinity precipitation of enzymes in the prior art. For example, lactate dehydrogenase and glutamate dehydrogenase have been precipitated with Bis-NAD; Analytical Biochemistry 133, pp. 409-416 (1983). Bis-NAD was used to precipitate both lactate dehydrogenase and glutamate dehydrogenase whereas yeast alcohol dehydrogenase required the presence of salt to enhance its precipitation; liver alcohol dehydrogenase did not precipitate. A Procion Blue analog of NAD was used to precipitate rabbit muscle lactate hydrogenase; ibid, 158, 382-389 (1986). Bis-Cibacron Blue has been used to precipitate 90% of lactate dehydrogenase, 50% bovine serum albumin, and 20% chymosin from solution; Journal of Chromatography, 376, 157-161 (1986). The ability of salts of metals such as zinc and copper to precipitate proteins is known. None of the prior art descriptions of protein precipitation, however, suggest the use of chelated metals for this purpose.